生物医药

The field of nucleic acid-based testing experienced a decade-long stagnation since the development of quantitative polymerase chain reaction (qPCR) in 1992 and isothermal amplification methods in the early 2000s. However, in 2016, the discovery of trans-nuclease activity in CRISPR-Cas systems revolutionized the molecular diagnostics for nucleic acids. A typical CRISPR diagnostic workflow comprises three phases: (1) target recognition through CRISPR RNA (crRNA)-guided hybridization; (2) signal transduction via trans-cleavage of engineered reporters (e.g., fluorophore-quencher oligonucleotides), and (3) signal readout using fluorescence, electrochemical, or colorimetric platforms. Emerging shortly prior to the COVID-19 pandemic, CRISPR diagnostics quickly gained prominence as a field-deployable alternative to qPCR due to its rapidity (<&#x2009;1&#xa0;h), minimal equipment requirements, and field adaptability. This technological paradigm underwent rigorous validation and refinement alongside the rapid evolution of SARS-CoV-2 detection, which facilitated its adaptation for cancer diagnosis. Recent advancements in sensitivity (attomolar-level detection) and specificity (single-nucleotide discrimination) have enabled transformative applications in cancer diagnostics, including: (1) identification of nucleic acid biomarkers, such as high-frequency somatic mutations, circulating nucleic acids and miRNAs; and (2) detection of non-nucleic acid biomarkers, including epigenetic aberrations, proteins, small molecules and metabolite biomarkers. This review chronicles the decadal evolution of CRISPR diagnostics, with particular emphasis on recent advancements of its application in cancer diagnosis. We critically evaluate persistent technical limitations, including PAM sequence restriction, suboptimal sensitivity and specificity, quantitative constraints, and unmet point-of-care testing (POCT) in complex biological matrices. Additionally, we discuss prospective solutions to address these challenges.

Over the past decade, CRISPR-based technologies have revolutionized our capacity to manipulate genomes, thereby reshaping the landscape of functional genomics research. Among the CRISPR toolkit, CRISPR/Cas9-mediated homology-directed repair (HDR) enables precise genome editing with predefined mutations, rendering it an indispensable tool for gene functional analysis, disease model construction, and the development of gene therapy strategies. Nevertheless, despite the robust efficiency of CRISPR/Cas9 in mediating gene knockouts, HDR-dependent gene knock-in (KI) remains a major bottleneck due to its inherently low efficiency. Herein, we report that the co-expression of PALB2 with the CRISPR/Cas9 nuclease could trigger an enhanced HDR effect. Specifically, the fusion of Cas9 with PALB2 elevated KI efficiency by approximately 1.7-fold in human HEK293T cells. Furthermore, this Cas9-PALB2 fusion strategy exhibited cross-cell-type efficacy, demonstrating its broad applicability beyond a single cell line. Notably, the combined application of the Cas9-PALB2 fusion system and Nocodazole further boosted KI efficiency to a remarkable 25.5%. Collectively, these findings establish the Cas9-PALB2 fusion as a highly potent and versatile strategy to augment HDR-mediated KI efficiency, highlighting its substantial potential for widespread utilization in applications that demand high-fidelity genome editing.

Synthetic gene-regulation logic is established in immortalized cell lines but remains largely aspirational in human induced pluripotent stem cells (hiPSCs) and derivatives. This gap constrains both mechanistic discovery and translational engineering in physiologically relevant models. We developed CIRI ( C ombinatorial I nducible C R ISPR in I PSCs), an isogenic, safe-harbor-engineered platform in which tetracycline-responsive single guide RNAs (sgR-NAs) carry modular RNA aptamers that recruit RNA-binding proteins and effector domains. This design enables multimodal regulation from a single catalytically inactive Cas9 (dCas9), exemplified by orthogonal CRISPR activation and interference (CRISPRa/i). After optimizing sgRNA-aptamer architectures, we achieved robust CRISPRa and CRISPRi in hiPSCs and hiPSC-derived cardiac organoids. CIRI rapidly channels hiPSC forward programming into skeletal myocytes by activating MYOD1 while repressing NANOG , POU5F1/OCT4 , and SOX2 . Combinatorial pooled dual-guide single-cell RNA sequencing screens identify ID3 as a road-block and KDM6B and SMARCD3 as synergistic enhancers of myogenic maturation. Together, CIRI establishes a programmable synthetic biology framework in human stem cell models.

Breast cancer (BC) is the most prevalent cancer among women and the second leading cause of cancer-related deaths globally, after lung cancer. Despite advances in treatment, BC remains a major contributor to cancer mortality worldwide, underscoring the need for innovative therapeutic approaches. The TopBP1 (DNA Topoisomerase II Binding Protein 1) gene, involved in DNA damage response and cell cycle regulation, has been associated with cancer progression and resistance to chemotherapy. This study investigates the potential of using CRISPR/Cas9 technology to knockout the TopBP1 gene as a novel strategy in breast cancer research. A pair of guide RNAs (gRNAs) was specifically designed to target the TopBP1 gene, inducing the deletion of exon 4. These gRNAs were transfected into the MCF7 breast cancer cell line, and the efficacy of genomic editing was validated using PCR and Sanger sequencing. Subsequent analyses employing real-time PCR and Western blotting were conducted to investigate the downstream effects of this genetic modification on gene expression. The CRISPR/Cas9 system successfully knocked out exon 4 of the TopBP1 gene in MCF7 breast cancer cells, as validated by PCR and Sanger sequencing. Real-time PCR analysis revealed a significant increase in Bax expression and a decrease in Bcl-2 expression in the knockout cells compared to controls. These changes indicate enhanced apoptotic activity following TopBP1 knockout, suggesting that MCF7 cells may become more sensitive to apoptosis. Overall, the findings support the hypothesis that targeting TopBP1 could play a critical role in promoting cell death in breast cancer, potentially offering a new therapeutic strategy. This study successfully employed the CRISPR/Cas9 system to knockout exon 4 of the TopBP1 gene in MCF7 breast cancer cells, resulting in reduced TopBP1 expression. The subsequent increase in the pro-apoptotic Bax gene and decrease in the anti-apoptotic Bcl-2 gene suggest that targeting TopBP1 could enhance apoptosis in breast cancer cells, offering a promising alternative to conventional treatments. Further research is necessary to fully explore the therapeutic potential of this approach.

Malaria caused by Plasmodium remains a major global health burden, especially in resource-limited settings where rapid and accurate diagnosis is essential. Most molecular diagnostic methods require DNA extraction, thermal cycling, and specialized laboratory infrastructure, limiting field applicability. We aimed to develop an extraction-free, one-pot CRISPR-based platform for rapid Plasmodium detection directly from whole blood under ambient conditions. Crude blood lysates were directly applied to a one-pot recombinase polymerase amplification (RPA)-CRISPR assay coupled with lateral flow detection. Analytical sensitivity and specificity were evaluated using purified DNA and whole-blood lysates. Diagnostic performance was evaluated using 116 malaria-positive specimens and 109 malaria-negative controls for the detection of Plasmodium falciparum and Plasmodium vivax, the two species responsible for most global malaria cases. The extract-free RPA-CRISPR assay achieved a detection limit of 100 copies/&#x3bc;L from crude blood lysates, corresponding to approximately 12-20 parasites/&#x3bc;L. The assay successfully detected both P. falciparum and P. vivax without cross-reactivity. Clinical evaluation showed 93.1% sensitivity and 100% specificity compared with reference qPCR. Sensitivity reached 97.8% for high-density infections, 95.1% for moderate-density infections, and 82.8% for low-density infections. The workflow was completed within 40&#x202f;min at room temperature without specialized instrumentation. This extraction-free, one-pot ambient-temperature platform enables rapid and sensitive detection of Plasmodium directly from whole blood. Although validated only for P. falciparum and P. vivax, the assay targets conserved Plasmodium 18S rRNA regions and may have broader applicability following further validation.

The generation of isogenic knockout (KO) cell lines for intracellular proteins using non-viral CRISPR-Cas9 approaches has long been technically demanding and time-consuming. Here, we describe a streamlined and cost-effective method based on ptARgenOM, an all-in-one mammalian expression vector designed for efficient delivery of the CRISPR-Cas9 system. This vector co-expresses the guide RNA (gRNA) and Cas9 endonuclease, which is fused to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein (EGFP) and the puromycin N-acetyltransferase. This design enables transient, expression-dependent antibiotic selection and fluorescence-based enrichment of successfully transfected cells, facilitating the rapid generation of isogenic KO populations or clones. The method is particularly well-suited, though not limited, to functional studies involving intracellular components of the cell death machinery, including both the extrinsic and intrinsic apoptotic signaling pathways. We illustrate the utility of this system by targeting and deleting FADD, Caspase-8, and RIPK1. This approach can be easily adapted to any intracellular target protein, offering a robust platform for gene function analysis in mammalian cells.

Precision agriculture is currently limited by a reliance on viral load quantification, a static metric that obscures the dynamic molecular arms race between invading pathogens and host immunity. To overcome this limitation, we present a multidimensional surface-enhanced Raman spectroscopy (SERS) platform designed to map the real-time topology of the host-virus interactome. Overcoming the spectral constraints of conventional assays, we engineered a de novo library of isomeric Raman reporters via rational positional and electronic tuning, enabling high-density spectral coding. Furthermore, by integrating CRISPR-dCas9 as an isothermal recognition module, we ensure multiplexed signals accurately reflect the stoichiometric integrity of viral and host gene expression due to the elimination of thermodynamic biases of traditional DNA hybridization. Applying this platform to the Nicotiana benthamiana-Potato virus Y (PVY) pathosystem, we dissect the longitudinal evolution of infection under ningnanmycin (NNM) treatment. Beyond enabling presymptomatic diagnosis at Day 1, our interactome analysis reveals that chronic infection stabilizes into a regulatory triangle clamped by the Vpg-PUB4 interface, whereas therapeutic relapse manifests as a chaotic, Vpg-centralized network. This work establishes interactome topology as a superior diagnostic metric to viral load, providing a blueprint for identifying drug resistance mechanisms and targeting the specific protein-protein interactions that drive viral persistence.

Cordyceps militaris, a renowned edible mushroom, produces orange-yellow fruiting bodies (FBs), primarily due to carotenoid accumulation. However, genetic mechanisms and functional roles underlying carotenoid biosynthesis remain poorly understood. Here, we identified Cmpks1, a light-induced gene encoding a reducing type I polyketide synthase, as a key regulator of pigment biosynthesis. Transcription of Cmpks1 was CmWC-1-dependent and upregulated during FB development. CRISPR/Cas9-mediated loss-of-function mutants of Cmpks1 exhibited stable albino phenotypes but retained FB differentiation. In addition to abolishing carotenoid biosynthesis, the disruption of Cmpks1 increased sensitivity to high light and oxidative stress, indicating its role in redox homeostasis. Metabolomic profiling of the &#x394;Cmpks1 mutant, including significantly reduced ergothioneine and elevated cordycepin, revealed extensive metabolic reprogramming, coupled with activation of compensatory survival mechanisms. These findings elucidate the genetic mechanisms governing pigment formation that influence the quality of Cordyceps products, offering new insights into the role of metabolites in fungal morphogenesis and stress adaptation.

CRISPR interference screens use catalytically inactive dCas9 fused to a repressor domain to enable genetic perturbations at the transcriptomic level. Interpretation of results involves identification of guide RNAs associated with the screen phenotype, followed by secondary analysis. During validation of a genetic screen, we observed different phenotypes from non-overlapping guide RNAs targeting one gene. Here, we developed POCKET-seq to map the binding of dCas9 genome-wide. We show that off-target binding occurs frequently and can generate false-positive interactions when it occurs near the promoter of genes associated with the screen phenotype. POCKET-seq classifies these false-positive and true-positive interactions using gene ontology.

Spatially resolved CRISPR screening in vivo has been limited to small perturbation panels and subsets of protein-coding RNAs. We present Perturb-DBiT, a method for co-sequencing of spatial total RNA whole transcriptomes and single guide RNAs (sgRNAs) on the same tissue section in situ. In a human cancer metastatic colonization model, we applied large (80,000+) sgRNA panels across tumor colonies in multiple consecutive tissue sections alongside their corresponding total RNA transcriptomes. We linked perturbations affecting long noncoding RNA covariation, microRNA-mRNA interactions and distinct amino acid-specific tRNA alterations to tumor migration and growth. By integrating transcriptional pseudotime trajectories, we further observed the impact of perturbations on clonal dynamics and cooperation. In an immune-competent syngeneic mouse model, investigation of the tumor immune microenvironment indicated distinct, synergistic effects on immune infiltration and suppression. Perturb-DBiT provides a spatially resolved comprehensive view of perturbation responses in complex tissues, including small and large RNA regulation, tumor proliferation, migration, metastasis and immune interactions.

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CRISPR-associated transposons (CASTs) couple target recognition to the insertion of large DNA cargoes, but how distinct targeting pathways are converted into productive and directional integration remains poorly understood 1 . Here we define the assembly pathway of a type I-B1 CAST from Anabaena variabilis , a system closely related to prototypical Tn7 that retains TnsD-mediated glmS recognition while incorporating CRISPR-based RNA-guided targeting 2 . Cryo-electron microscopy structures across multiple intermediates define a structural trajectory from two-step TnsD-mediated target recognition and stepwise assembly of the AAA+ ATPase TnsC to recruitment and activation of the split TnsA/TnsB transposase module. This trajectory culminates in an asymmetric strand-transfer complex that provides a structural basis for insertion orientation and supports a role for ATP hydrolysis in productive transpososome assembly. In parallel, RNA-guided targeting structures refine the functional PAM to ATG, define TniQ recruitment by Cascade, and show how CRISPR-based recognition converges on the shared TnsABC machinery. Together, these findings establish DNA remodeling and minor-groove positioning of TnsC as a common structural signal that converts protein- and RNA-guided target recognition into directional DNA insertion.

2-Phenylethanol (2-PE) is a key aromatic alcohol contributing to the rose-like odor in brewed wines, primarily synthesized by yeast metabolism with a typical yield of less than 100 mg/L. To enhance the 2-PE content in brewed wines, this study used CRISPR-Cas9 gene editing technology to delete the ARO8 gene (encoding aromatic transaminase I) in Saccharomyces cerevisiae SY. The single-factor experiments were performed to optimize the fermentation process, and the 2-PE content in the brewed wine was measured by high-performance liquid chromatography. The results demonstrated that the 2-PE content in whisky fermented by the SY-A8 was 0.73 g/L, increasing 23.73% compared to SY. The fermentation conditions of SY-A8 were optimized through single-factor experiments and the Box-Behnken design. The optimal conditions were a sugar concentration of 46.30 g/L, a fermentation time of 6 days, and an L-phenylalanine concentration of 1.43 g/L. The high 2-phenylethanol aroma whisky was brewed with a higher 2-phenylethanol content of 3.68 g/L in a 1 L fermenter at the optimal conditions. In conclusion, the modification of Saccharomyces cerevisiae by CRISPR-Cas9 gene editing combined with fermentation process optimization provides an effective technical strategy for improving the 2-PE content in whisky, thereby providing a research perspective for the flavor enhancement of whisky and other brewed wines.

Hulunbuir short-tailed sheep (HSTS) and Hu sheep (HS) exhibit distinct tail phenotypes linked to ecological adaptation, with HSTS carrying a loss-of-function mutation (c.G334T) in the T gene while HS retain the wild-type allele. However, the cellular and molecular mechanisms underlying T-mediated tail development remain unclear. Here, we performed single-cell RNA sequencing on HSTS and HS embryos at embryonic days 16 and 19 (E16 and E19), complemented by cross-species validation using a CRISPR/Cas9 mouse model carrying the same mutation. We identified 12 cell types in E16 HSTS and E16 HS embryos, and 15 cell types in E19 HSTS and E19 HS embryos and found that the MDK_ITGA6+ITGB1 ligand-receptor pair consistently mediated core intercellular communication. The MDK_ITGA6+ITGB1 axis mediates intercellular communication critical for tail bud formation; BMP activation and FGF repression disrupt AER survival, leading to tail shortening. Developmental trajectories showed a shift from early progenitor states at E16 to terminal differentiation at E19. Crucially, HSTS embryos showed transcriptomic signatures consistent with premature AER regression. The T mutation showed transcriptomic signatures of increased BMP pathway activity and reduced FGF8 expression, which may disrupt AER survival and contribute to the short-tail phenotype. In the mouse model, mutant T expression was reduced, and expression dynamics of WNT5B and FGF8 were perturbed, corroborating the sheep findings; however, homozygous T mutation causes embryonic lethality in mice but not in sheep, indicating species-specific differences. This study provides single-cell transcriptomic evidence linking the T c.G334T mutation to premature AER regression in sheep, complemented by cross-species validation in a CRISPR/Cas9 mouse model, offering new insights into the cellular mechanisms of tail development and may provide a basis for future investigations into tail-related breeding markers, pending experimental validation. These changes are associated with AER maintenance and tail outgrowth.

Astrocytes play essential roles in neuronal development, function, and disease, yet existing methods to derive astrocytes from human pluripotent stem cells (hPSCs) are complex and can involve months of in vitro maturation. We developed a genomic safe-harbor knock-in system for inducible expression of the astrogenic transcription factors NFIA, NFIB, and SOX9, enabling rapid and robust generation of functional induced astrocytes (iAstrocytes). Across five hPSC lines, NFIB-SOX9 and NFIA-NFIB-SOX9 combinations efficiently generated highly pure populations expressing astrocyte-specific and synaptogenic genes. iAstrocytes displayed cytokine-induced expression of complement factors C3 and C4 and were amenable to CRISPR interference (CRISPRi) gene expression knockdown. Optimization of culture conditions enabled survival of NFIB-SOX9 iAstrocytes in co-culture with human induced neurons (iNeurons). Through pharmacological and genetic perturbations, we uncovered a previously undescribed phenomenon in which co-culture with iAstrocytes promoted the development of synchronized iNeuron network calcium activity mediated by specific gap junction proteins. This rapid and genetically tractable iAstrocyte platform provides a robust model to dissect human genetic and environmental effects on astrocyte-neuron interactions.

Erwinia amylovora, the causative agent of fire blight, poses a significant threat to global pome fruit production. This study presents a comprehensive genomic analysis of 317 E. amylovora strains and 227 Erwinia phages to elucidate virulence evolution, phage-host dynamics, and the genomic signatures of the co-evolutionary arms race. Our analysis suggests that a substantial portion of E. amylovora's virulence factors (VFs) share evolutionary origins with diverse plant, human, and animal pathogens, underscoring widespread horizontal gene transfer. We identified bacterial phage hydrolases&#x2011;like proteins that share phylogenetic and domain-level similarities with phage endolysins. These observations are consistent with the possibility that some bacterial hydrolases originated from phage-derived ancestors, although functional repurposing remains to be experimentally validated. Crucially, our analysis identifies systematic, non-random associations between bacterial defense systems (e.g., RM, CRISPR-Cas, TA) and mobile anti-defense genes. Statistical correlations show strong patterns of co-occurrence and mutual exclusivity, which are consistent with an ongoing phage-bacteria arms race. These patterns provide a genomic basis for generating hypotheses about co-evolutionary dynamics. These findings may advance our understanding of E. amylovora pathogenicity and phage interactions, offering foundational insights for developing targeted phage-based biocontrol strategies against this devastating plant pathogen. Experimental validation of the predicted virulence factors and defense correlations is warranted to confirm their biological roles.

Insulin-like growth factor 1 (IGF-1) signaling plays a complementary role to insulin signaling in glucose metabolism homeostasis. This study characterized the physiological roles of the IGF1 receptor A (Igf1ra) and B (Igf1rb) in zebrafish. The transcripts of igf1ra and igf1rb were detected in multiple zebrafish tissues, including the liver, muscle, and brain. Zebrafish lacking igf1ra or igf1rb were generated using CRISPR/Cas9 technology. Both igf1ra-/- and igf1rb-/- zebrafish exhibited stunted growth. Reduced BMI was found in igf1ra-/- zebrafish, while BMI increased in igf1rb-/- zebrafish. Hyperglycemia and increased hepatic glycogen were observed in igf1ra-/- zebrafish, while blood glucose levels in igf1rb-/- zebrafish were normal. No significant difference in whole-body or hepatic triglyceride content was observed in igf1ra-/- zebrafish, while the whole-body and hepatic triglyceride content of igf1rb-/- zebrafish increased compared to their wild-type control siblings. Further analyses of the expression patterns of key genes involved in glucose and lipid metabolism were conducted on igf1r mutants. Decreased levels of genes involved in glucose absorption and glycolysis and increased levels of genes involved in gluconeogenesis and glycogen synthesis were observed in igf1ra-/- zebrafish, but not in igf1rb-/- zebrafish. Conversely, significantly decreased levels of transcripts involved in lipolysis and increased levels of transcripts involved in the lipogenesis process were observed in igf1rb-/- zebrafish, but not in igf1ra-/- zebrafish. Restricted cell growth and protein synthesis signaling, including AKT and mTOR activation, was also detected in igf1ra-/- zebrafish, while a moderate elevation in AKT and mTOR activity was seen in igf1rb-/- zebrafish. Taken together, our results suggest that functional divergence occurred after the duplication of the zebrafish igf1r gene, with igf1ra primarily modulating glucose absorption and utilization, and igf1rb primarily affecting lipid metabolism in the somatotropic axis.

To overcome limitations in traditional case-based teaching, this study developed an AI-driven workflow using DeepSeek to generate contemporary, interdisciplinary clinical biochemistry cases. Through human-AI collaboration, eight structured cases covering key topics such as carbohydrate and lipid metabolism were created, each including a clinical description, molecular mechanisms, and Q&A, followed by instructor review. Fifteen medical students and 15 instructors evaluated the cases using a 5-point Likert scale across multiple dimensions, while student performance was compared between a group using AI-generated cases and a control group. The AI generated each case in 10-15&#x2009;min, significantly faster than manual development. Cases presented a logical progression from molecular mechanism to clinical management and incorporated recent advances such as CRISPR and PCSK9 inhibitors. Content integration received the highest ratings, though instructors scored pedagogical applicability lower. Error analysis indicated that AI excelled in maintaining logical consistency, whereas human reviewers enhanced precision in clinical details. Students who used the AI-generated cases achieved significantly higher examination scores than the control group (p&#x2009;<&#x2009;0.05). In conclusion, DeepSeek-generated cases are efficient, interdisciplinary, and innovative. Human review remains essential for ensuring clinical rigor, particularly in nuanced scenarios. This collaborative approach enhances both the efficiency of case development and the educational quality of biochemistry teaching materials.

Ensuring global food security under accelerating climate change requires transformative approaches to crop improvement that extend beyond the limits of traditional breeding and gene editing. While domestication and modern agriculture have delivered substantial gains in productivity, these advances often came at the cost of genetic diversity, stress resilience, and developmental plasticity. Plants, however, inherently exhibit remarkable flexibility in their morphology and development, as evidenced by the vast diversity of organ shapes, cell types, and adaptive responses that have evolved across lineages. This natural design space provides a foundation for reimagining plant architecture using synthetic biology. Recent advances in plant synthetic biology, including programmable transcription factors, CRISPR-based regulatory systems, synthetic gene circuits, orthogonal signalling pathways, and plant artificial chromosomes, now enable precise, modular, and environmentally responsive manipulation of developmental processes. These tools allow researchers to rewire hormone pathways, tune quantitative gene expression, integrate multiple environmental signals, and create novel regulatory modules that operate independently of endogenous networks. Beyond understanding plant development, these capabilities open avenues for engineering crops with dynamic architectures, enhanced plasticity, and improved resilience to complex and fluctuating stresses. In this review, we synthesise insights from natural diversity, developmental biology, and synthetic regulatory engineering to outline how plant architecture can be rationally redesigned. We argue that integrating synthetic biology with modern breeding and modelling frameworks will be essential for generating the next generation of programmable crops; i.e., varieties capable of sustaining productivity and stability in an era of unprecedented environmental and geopolitical changes.

What fraction of the human genome is essential for cellular viability? To date, essentiality in human cells has been mapped almost exclusively at the level of individual open reading frames (ORFs). Whether noncoding regions and broader architectural features of the genome are required for human cells to remain viable, and where the boundaries of any such regions lie, remains largely unexplored. Here we introduce Shred-seq, which couples Type I-C CRISPR-Cas3-mediated deletions with phage polymerase-based genotyping to enable large-scale deletion scans of the human genome. Shred-seq leverages thousands of genomically integrated, mapped target sites as launchpads for Cas3 to initiate unidirectional deletions ranging in size from hundreds of base pairs (bp) to hundreds of kb. Breakpoints are directly captured at high resolution by in vitro or in situ transcription from flanking phage polymerase promoters, enabling bulk or single-cell phenotyping, respectively. In this proof-of-concept, we generate and genotype 36,257 independent deletions originating from 9,604 Cas3 launchpads, individually spanning 100 bp to 500 kb, collectively covering 461 Mb (14% of the human genome), and totaling to 2.55 Gb of deleted sequence (&#x223c;10-fold coverage of these regions). Surviving deletions are depleted not only for essential protein-coding genes, but also for active, conserved and mutation-constrained non-coding sequences, directly quantifying purifying selection across both coding and noncoding intervals. Deletion length distributions further enable annotation-agnostic fine mapping of essential region boundaries and establish an empirical lower bound on the fraction of the human genome required for cellular viability. Finally, we demonstrate compatibility with single-cell RNA-seq (scRNA-seq), enabling direct linkage of specific deletions to transcriptional phenotypes. Together, Shred-seq provides a scalable platform for the systematic dissection of genome architecture and noncoding function, as well as for generating training data for predictive and generative models of genome structure-function, analogous to the roles that conventional Perturb-seq screens play for ORFs. By enabling the empirical delimitation of the genomic content required for human cellular viability, Shred-seq may also open a path to the construction of a minimal human genome.